Rapamycin derivative

ABSTRACT

A compound of formula (I). ##STR1##

This application is a 371 of PCT/GB92/02235, filed Dec. 1, 1992.

The present invention relates to a novel compound and derivativesthereof, to processes for their production, to pharmaceuticalformulations containing them, to their use in medical therapy,particularly in the treatment of bacterial and fungal infections, andalso to their use as immunosuppressants and in treating carcinogenictumours. Rapamycin is a known compound and is a member of the trieneclass of antibiotics. It was first isolated as an extract of thebacterium Streptomyces hygroscopicus and reported to have antifungalactivity (British Patent 1436447). Subsequently rapamycin has beenimplicated as an immunosuppressant (Martel R. R. et al Can. J. Physiol.Pharmacol. 55, 48-51, 1977). At least one rapamycin-producing strain ofStreptomyces hygroscopicus was deposited with the Northern Utilizationand Research Division, Agricultural Research Service, U.S. Department ofAgriculture, Peoria, Ill., U.S.A under accession number NRRL 5491.Rapamycin, and methods for its preparation by culturing NRRL 5491 aredisclosed by U.S. Pat. No. 3,929,992 issued Dec. 30, 1975, the entiredisclosure of which is hereby incorporated by reference.

A large number of microorganisms have been found to produce a variety ofmetabolites which have subsequently been isolated and have been shown topossess useful therapeutic properties. One such compound is 14-methylenerapamycin. This is believed to be a novel compound and has been found tohave useful antifungal activity, anticancer activity and alsoimmunomodulatory properties.

Accordingly the present invention provides a compound of the formula (I)which is believed to have the following structure: ##STR2##

This novel compound has been found to have useful antimicrobialanticancer and immunomodulatory activity.

This compound is referred to as 14-methylene rapamycin according to thenumbering system of J. Findlay et al., Can. J. Chem. (1980) 58, 579,however according to the more recent numbering system of J. McAlpine etal., J. Antibiotics (1991) 44, 688 this would be known as 9-methylenerapamycin.

The effect of the differing nomenclatures on the numbering of formula Iis shown in formulas II and III below. Formula II represents14-methylene rapamycin in one of its isomeric forms and formula III9-methylene rapamycin. ##STR3##

The compound could alternatively be referred to as 14-desoxorapamycin.

For simplicity, 14-methylene rapamycin is referred to in the followingtext, however where relevant both systems are also referred to.

The invention in a second aspect, further provides a process for theproduction of 14-methylene rapamycin which comprises cultivating aproducing microorganism and subsequently isolating 14-methylenerapamycin or derivatives thereof.

The compound of the present invention has the following characteristics:

i) it has an apparent molecular weight of 899 by Fast Atom Bombardment(FAB) Mass spectroscopy,

ii) it may be obtained by the cultivation of a microorganism from thegenus Streptomyces,

iii) ¹³ C NMR spectroscopy reveals 51 carbons in the molecule,

iv) it has a characteristic UV spectrum with peaks at 268, 277 and 289nm,

v) it is useful as an antifungal agent,

vi) it is useful as an immunomodulatory agent.

The compound of the present invention may be obtained by the cultivationof a producing organism and the recovery of it or a derivative thereoffrom the culture.

The term `cultivation` (and derivatives of that term) as used hereinmeans the deliberate aerobic growth of an organism in the presence ofassimilable sources of carbon, nitrogen, sulphur and mineral salts. Suchaerobic growth may take place in a solid or semi-solid nutritive medium,or in a liquid medium in which the nutrients are dissolved or suspended.The cultivation may take place on an aerobic surface or by submergedculture. The nutritive medium may be composed of complex nutrients ormay be chemically defined.

It has been found that suitable microorganisms for use in thecultivation process according to the invention include bacterial strainsbelonging to the genus Streptomyces which are capable of elaborating14-methylene rapamycin. It has further been found that an example ofsuch a strain is sp. NCIB 40319 and also mutants thereof, which has beenisolated from nature.

The term `mutant` as used herein includes any mutant strain which arisesspontaneously or through the effect of an external agent whether thatagent is applied deliberately or otherwise. Suitable methods ofproducing mutant strains including those outlined by H. I. Adler in`Techniques for the Development of Microorganisms` in `Radiation andRadioisotopes for Industrial Microorganisms`, Proceedings of aSymposium, Vienna, 1973, page 241, International Atomic EnergyAuthority, and these include:

(i) Ionizing radiation (e.g. X-rays and gamma-rays), u.v. light, u.v.light plus a photosensitizing agent (e.g. 8-methoxypsoralen), nitrousacid, hydroxylamine, pyrimidine base analogues (e.g. 5-bromouracil),acridines, alkylating agents (e.g. mustard gas, ethyl-methanesulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and

(ii) Genetic techniques, including, for example, recombination,transformation, transduction, lysogenisation, lysogenic conversion,protoplast fusion and selective techniques for spontaneous mutants.

Using the methods of Becker B. Lechevalier M. P., Gordon R. E.,Lechevalier H. A., 1964, Appl. Microbiol. 12, 421-423 and Williams S.T., Goodfellow M, Wellington E. M. H., Vickers J. C., Alderson. G.,Sneath P. H. A., Sackin M. J., and Mortimer M. 1983 J. Gen. Microbiol.129, 1815-1830, Sp. NCIB 40319 has been identified as a new, strain ofStreptomyces hygroscopicus and therefore also forms a part of thepresent invention, particularly in biologically pure form. It has beendeposited at the National Collection of Industrial and Marine BacteriaLtd. (N.C.I.B), Aberdeen, Scotland under number 40319 on 14 Sep. 1990.

Strain NCIB 40319 has been characterised as follows:

The method of whole-cell amino acid analysis was that described byBecker et al (1964). Identification media used for the characterisationof the culture were as described by Williams et al (1983). In addition,starch casein agar (Waksman S. A., 1961. The Actinomycetes Vol. 2Williams and Wilkins Co. Baltimore pp 1-363) was used for themorphological description of the culture.

The microorganism was characterised by inoculating agar blocks from awell grown plate into Y broth (see Table 1 ) and incubating for threedays at 28° C. on a shaker. It was then centrifuged for 20 minutes at3660 rpm, washed twice with distilled water, then finally resuspended inphosphate buffered saline (Dulbecco A). This inoculum was plated ontomedia commonly used for the identification of members of theActinomycetales as above. Plates were incubated at 28° C. and theresults were read at varying times but most were commonly taken at 14days. The colours are described in common terminology but exact colourswere determined by comparison with colour chips from the MethuenHandbook of colour (3rd Edn).

Results:

Cell Wall analysis

The whole-cell hydrolysates contained LL-diaminopimelic acid. Theobservations of growth and appearance of the organism were as follows:

Yeast extract-Malt extract Agar (ISP 2 Difco)

Growth good, cream 2 2a), with a white powdery centre. Colonies raisedand rather wrinkled, no sporulation.

Inorganic Salts Starch Agar (ISP4 Difco)

Growth good, white with pale grey to grey (1 1b, 1 1c) aerial mycelium.Colonies quite flat with slightly raised centre. Reverse cream (2 2a).

Glycerol Asparagine Agar (S. A. Waksman, 1961, p328). medium No. 2.

Growth moderate to good, white with grey centre (1 1d). Colonies flat,reverse cream (2 2a).

Starch Mineral Salts Agar

Growth very poor, opaque small colonies. No aerial mycelium.

Starch Casein Agar

Growth good, white with light grey to grey central area (1 1c, 1 1d),occasional small patch of white non-sporulating mycelium in greysporulating areas. Tiny colourless droplets over the grey areas.Colonies fairly flat and gently rounded. Small black hygroscopic patchesmay occur after 4 weeks incubation.

Morphological Properties

These were observed after two weeks incubation on starch casein agar:spore mass in grey colour-series; spore chains in section spirales,tightly coiled or slightly open, of small diameter, generally 2-6 coils,occasionally more, may aggregate into hygroscopic masses. There was nofragmentation of vegetative mycelium.

Biochemical Properties

See Table 2 for full details. In summary, melanin not produced; nitratenot reduced to nitrite in organic nitrate broth; H₂ S produced inpeptone-yeast extract iron broth; no growth on inhibitors; degradationonly of arbutin, antibiosis only against Bacillus subtilis. Carbohydrateutilization glucose, cellobiose, fructose, inositol, mannitol,raffinose, rhamnose and xylose. Nitrogen sources used: asparagine,histidine and hydroxyproline, α-amino-butyric acid used only slightly.

Determination of Identification Scores

These were obtained using the Matiden program (Sneath P. H. A., 1979.Computers and Geosciences 5 195-213) which provides the bestidentification scores for known or unknown strains against the percentprobability matrix of Williams et al (1983). Willcox Probability--thenearer the score reaches 1.0, the better is the fit of an unknown with agroup in the matrix (scores of >0.85 acceptable) Taxonomic distance--lowscores indicate relatedness (scores <0.3 acceptable). The organism hadacceptable identification scores with cluster 32 (violaceoniger) whichcontains Streptomyces hygroscopicus species.

Conclusion:

The culture is characterised by the grey spores in mass, the negativemelanin reaction and the spores which are arranged in spirally coiledchains. The spore chains may coalesce into hygroscopic masses. Theculture utilised a wide range of carbohydrate sources. The whole-cellhydrolysate indicates the presence of LL-diaminopimelic acid.

                  TABLE 1                                                         ______________________________________                                        1.     Y broth                g/L                                             ______________________________________                                        Special peptone (Oxoid)   2.5                                                 Lab Lemco powder (Oxoid)  2.5                                                 Tryptone (Oxoid)          2.5                                                 Neutralized soya peptone (Oxoid)                                                                        2.5                                                 Starch (BDH)              2.5                                                 Glucose (BDH)             2.5                                                 Malt Extract (Oxoid)      2.5                                                 Glycerol (Fisons)         2.5                                                 CaCl.sub.2.2H.sub.2 O (BDH)                                                                             0.05                                                MgCl.sub.2.6H.sub.2 O (Sigma)                                                                           0.05                                                NaCl (BDH)                0.05                                                FeCl.sub.3 (Sigma)        0.015                                               ZnCl.sub.2 (Sigma)        0.0025                                              CuCl.sub.2.2H.sub.2 O (Sigma)                                                                           0.0025                                              MnSo.sub.4.4H.sub.2 O (Sigma)                                                                           0.0025                                              CoCl.sub.2.6H.sub.2 O (BDH)                                                                             0.025                                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Biochemical Characteristics of NCIB 40319                                     Test           Result                                                         ______________________________________                                        Melanin production                                                                           -                                                              Use of Carbohydrates:                                                         Adonitol       -                                                              Cellobiose     +                                                              D-Fructose     +                                                              Meso-Inositol  +                                                              Inulin         -                                                              Mannitol       +                                                              Raffinose      +                                                              L-Rhamnose     + Willcox Probability                                          D-Xylose       +                                                              D-Glucose      + Cluster 32 violaceoniger = 0.936                             Use of Nitrogen sources:                                                      DL-α-Aminobutyric Acid                                                                 +/-                                                            L-Histidine    + Taxonomic Difference                                         L-Hydroxyproline                                                                             +                                                              Asparagine     + Cluster 32 violaceoniger = 0.284                             Degradation of:                                                               Allantoin      -                                                              Arbutin        +                                                              Xanthine       -                                                              Pectin         -                                                              Lecithin       -                                                              Nitrate Reduction                                                                            -                                                              H.sub.2 S Production                                                                         +                                                              Growth on Inhibitors:                                                         Sodium azide (0.01% w/v)                                                                     -                                                              NaCl (7.0% w/v)                                                                              -                                                              Phenol (0.1% w/v)                                                                            -                                                              Growth at 45° C.                                                                      -                                                              Antibiosis to:                                                                Aspergillus niger                                                                            -                                                              Bacillus subtilis                                                                            +                                                              Streptomyces murinus                                                                         -                                                              ______________________________________                                    

The fermentation medium for cultivating sp. NCIB 40319 suitably containssources of assimilable carbon and assimilable nitrogen together withinorganic salts. Suitable sources of nitrogen include yeast extract,soyabean flour, meat extract, cottonseed, flour, malt, distillers driedsolubles, amino acids, protein hydrolysates and ammonium and nitratenitrogen. Suitable carbon sources include glucose, lactose, maltose,starch and glycerol. Suitably the culture medium also includes alkalimetal ions (for example, sodium), halogen ions (for example, chloride),and alkaline earth metal ions (for example calcium and magnesium), aswell as trace elements such as iron and cobalt.

The cultivation may suitably be effected at a temperature of about 20°to 35° C., advantageously 20° to 30° C., and the culture may suitably beharvested up to 7 days, advantageously about 3 to 5 days, after theinitiation of fermentation in order to give an optimum yield of thedesired product.

The desired product or a derivative thereof may then be isolated fromthe culture medium and worked up and purified using conventionaltechniques for such compounds. All such isolation and purificationprocedures may conveniently be effected at cool to ambient temperature,for example at a temperature within the range of from 4° to 40° C.,conveniently from 20° to 35° C.

The desired compound may readily be identified in a routine manner bytesting for antifungal activity and/or by monitoring the h.p.l.c.retention time.

Suitably, the separation procedure may include a high-performance liquidchromatography step, preferably as the last step. Elution may beeffected using aqueous methanol.

14-methylene rapamycin may be crystalline or non-crystalline and, ifcrystalline, may optionally be hydrated or solvated.

The compounds of the present invention can exist in free form or, whereappropriate, in salt form. Pharmaceutically acceptable salts andpreparation are well-known to those of skill in the art. Thepharmaceutically acceptable salts of the compounds of this inventioninclude the conventional non-toxic salts or the quaternary ammoniumsalts of such compounds which are formed, for example, from inorganic ororganic acids of bases.

The compounds according to the invention are suitably provided insubstantially pure form, for example at least 50% pure, suitably atleast 60% pure, advantageously at least 75% pure, preferably at least85% pure, more preferably at least 95% pure, especially at least 98%pure, all percentages being calculated as weight/weight. An impure orless pure form of a compound according to the invention may, forexample, be used in the preparation of a more pure form of the samecompound or of a related compound (for example a correspondingderivative) suitable for pharmaceutical use.

14-methylene rapamycin has antifungal, anticancer and immunosuppressantproperties and are useful for the treatment of fungal infections inanimals, especially mammals, including humans, in particular humans anddomesticated animals (including farm animals). Examples of pathogenicfungi include, but are not limited to Candida albicans and other candidaspecies, Microsporum gypsum, Trichophyton mentagophytes, Aspergillus spand Sporotrichum sp. The ability of the compound to inhibit the growthof pathogenic fungi may be demonstrated or predicted by standard testsknown and used for this purpose, for example the yeast test described inthe Examples. The compound may be used for the treatment of topicalfungal infections in man caused by, among other organisms, species ofCandida, Trichophyton, Microsporum or Epidermophyton or in mucosalinfections caused by Candida albicans (e.g. thrush and vaginalcandidiasis). It may also be used in the treatment of systemic fungalinfections caused by, for example Candida albicans, Cryptococcusneoformans, Aspergillus fumigatus, Coccidiodes, Paracocciciodes,Histoplasma or Blastomyces spp. It may also be of use in treatingeumycotic mycetoma, chromoblastomycosis and phycomycosis.

The compound of the invention is active as an immunomodulatory agent.The term "immunomodulatory agent" means that the compound of theinvention is capable of inducing immune suppression by inhibiting T (andB) cell responses in vitro and/or by producing a statisticallysignificant decrease in the inflammation system response mediatedsecondary lesion in the adjuvant induced arthritis. The fact that thecompounds of this invention have utility in inducing immuno suppressionmeans that they are useful in the treatment or prevention of resistanceto or rejection of transplanted organs or tissues (eg kidney, heart,lung, bone marrow, skin, cornea, etc); the treatment or prevention ofautoimmune, inflammatory, proliferative and hyperproliferative diseases,and of cutaneous manifestations of immunologically mediated diseases (egrheumatoid arthritis, lupus erythematosus, systemic lupus erythematosus,Hashimotos thyroiditis, multiple sclerosis, myasthenia gravis, type 1diabetes, uveitis, nephrotic syndrome, psoriasis, atopical dermatitis,contact dermatitis and further eczematous dermatitides, seborrheicdermatitis, Lichen planus, Pemplugus, bullous Phemphigold, Epidermolysisbullosa, uritcaris, angiodemas, vasculitides, erythemas, cutaneouseosinophilias, Alopecia areata, etc.); the treatment of reversibleobstructive airways disease, intestinal inflammations and allergies (eg,Coeliac disease, proctitis, eosinophilia gastroenteritis, mastocytosis,Chrohn's disease and ulcerative colitis) and food related allergies (egmigraine, rhinitis, and eczema). Other indications for therapy using animmunomodulatory agent include, but are not limited to, the treatment ofthe following disease states: acute transplantation/graft rejection,progressive systemic sclerosis, multiple myeloma, atopic dermatitis,hyperimmunoglobulin E, hepatitis B antigen negative chronic activehepatitis, Familial Mediterranean fever, Grave's disease, autoimmunehemolytic anemia, primary biliary cirrhosis, inflammatory bowel diseaseand insulin dependent diabetes mellitus

Accordingly the invention provides 14-methylene rapamycin or aderivative thereof for use in medical therapy. Preferably for use as anantifungal agent or an anticancer agent or an immunomodulatory agent.

The invention further provides a method of treating a human or animalsuffering from a fungal infection by the administration of an effectiveamount of 14-methylene rapamycin or derivative thereof.

Moreover, the invention provides a method of treating a human or animalin need of immunomodulation by administration of an effective amount of14-methylene rapamycin or a derivative thereof.

The invention further provides a pharmaceutical composition comprising acompound of the formula (I) or a pharmaceutically acceptable saltthereof together with a pharmaceutically acceptable diluent or carrier.The composition is preferably for human use in tablet, capsule,injectable or cream form.

For human use, 14-methylene rapamycin or derivatives thereof can beadministered alone, but will generally be administered in admixture witha pharmaceutical carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. For example, theymay be administered orally in the form of a tablet containing suchexcipients as starch or lactose, or in a capsule or ovule either aloneor in admixture with excipients, or in the form of an elixir orsuspension containing a flavouring or colouring agent. They may beinjected parenterally, for example, intravenously, intramuscularly orsubcutaneously. For parenteral administration, they are best used in theform of a sterile solutions which may contain other substances, forexample, enough salts or glucose to make the solution isotonic.

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of compound would be forthe purposes of inhibiting pathogenic fungi growth. Generally, however,an effective dosage will be in the range of about 0.05 to 100 milligramsper kilogram body weight per day.

For oral and parenteral administration to human patients suffering froma fungal infection, it is expected that the daily dosage level of theantifungal compounds of formula (I) will be from 0.1 to 10 mg/kg (individed doses) when administered by either the oral or parenteral route.Thus tablets or capsules of the compounds can be expected to containfrom 5 mg to 0.5 g of active compound for administration singly or twoor more at a time as appropriate. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual patient and will vary with the age, weight and response ofthe particular patient. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of compound would be forthe purpose of inducing immunosuppression. Generally, however, aneffective dosage will be in the range of about 0.05 to 100 milligramsper kilogram body weight per day. For a human patient in need ofimmunomodulation the daily parenteral or oral dosage regimen for thecompound or derivative thereof will preferably be from 0.1 mg/kg to 30mg/kg.

The compounds of this invention should also be useful for treatingcarcinogenic tumors in a mammal. More specifically, the compounds shouldbe useful for reducing tumor size, inhibiting tumor growth and/orprolonging the survival time of tumor-bearing animals. Accordingly, thisinvention also relates to a method of treating carcinogenic tumors in ahuman or other animal comprising administering to such human or animalan effective, non-toxic amount of a compound of Formula II. One skilledin the art would be able, by routine experimentation, to determine whatan effective, non-toxic amount of compound would be for the purpose oftreating carcinogenic tumors. Generally, however, an effective dosage isexpected to be in the range of about 0.05 to 100 milligrams per kilogrambody weight per day.

The compounds and compositions according to the invention may beformulated for administration in any convenient way for use in human orveterinary medicine, by analogy with other antifungal, anticancer orimmunomodulatory agent.

The compounds and tablets and capsules for oral administration may be inunit dosage form, and may contain conventional excipients including, forexample, binding agents, for example, syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; and pharmaceuticallyacceptable wetting agents, for example sodium lauryl sulphate. Thetablets may be coated according to methods well known in normalpharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or anothersuitable vehicle before use. Such liquid preparations may containconventional additives, including, for example, suspending agents, forexample sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel orhydrogenated edible fats; emulsifying agents, for example lecithin,sorbitan monooleate or acacia; non-aqueous vehicles (which may includeedible oils), for example almond oil, oily esters (for exampleglycerides), propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid; and, ifdesired, conventional flavouring and colour agents.

Compositions according to the invention intended for topicaladministration may, for example, be in the form of ointments, creams,lotions, eye ointments, eye drops, ear drops, impregnated dressings, andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation. Compositions according to the invention may be formulatedas suppositories, which may contain conventional suppository bases, forexample cocoa-butter or other glycerides.

Compositions according to the invention intended for parenteraladministration may conveniently be in fluid unit dosage forms, which maybe prepared utilizing the compound and a sterile vehicle,propyleneglycol. The compound, depending on the vehicle andconcentration used, may be either suspended or dissolved in the vehicle.Parenteral suspensions may be prepared in substantially the same mannerexcept that the compound is suspended in the vehicle instead of beingdissolved and sterilisation cannot be accomplished by filtration. Thecompound may instead be sterilised by exposure to ethylene oxide beforebeing suspended in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in such suspensions in order to facilitateuniform distribution of the compound.

It will be recognised by the person skilled in the art that the optimalquantity and spacing of individual dosages of the compound of theinvention will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration and theparticular animal to be treated and that such optima can be determinedby conventional techniques.

The following examples serve to illustrate the present invention.

EXAMPLE 1 Preparation of 14-methylene rapamycin from a culture ofStreptomyces by fermentation

A culture producing 14-methylene rapamycin has been classified asStreptomyces sp. and has been deposited in the national Collection ofIndustrial and Marine Bacteria, 23, St. Machar Drive, Aberdeen AB2 1RY,Scotland, UK. under the accession number NCIB 40319.

The culture was isolated from a termite hill at Abuke, Gambia.

1.0 Fermentation

1.1 Inoculum Preparation

Sporulating cultures grown on starch/casein agar slopes [soluble starch(BDH Poole, Dorset), 10 g/l; casein (white soluble), 1 g/l; K₂ HPO₄, 0.5g/l; MgSO₄.7H₂ O, 0.5 g/l; agar technical (oxoid No.3, OxoidBasingstoke) 18 g/l] in McCartney bottles were treated with 5 ml 0.02%Tween 80 to produce a spore suspension. Each of two 500 ml flaskscontaining 100 ml RS1 medium [Oxoid neutralised Soy peptone, 10 g/l;glucose monohydrate, 20 g/l; bakers yeast, 5 g/l; NaCl, 2 g/l; ZnSO₄.7H₂O, 0.05 g/l; MgSO₄.7H₂ O, 0.125 g/l; MnSO₄.4H₂ O, 0.01 g/l; FeSO₄.7H₂ O,0.02 g/l adjusted to pH 7.0 with 5N sodium hydroxide] were inoculatedwith 2 ml spore suspension. Flasks were incubated at 25° C., 240 rpm (50mm throw) for 72 hours.

After 72 hours the contents of the 2 flasks were pooled and 16 mlaliquots of the pooled culture used to inoculate each of ten×2 literflasks each containing 400 ml RS1 medium. Flasks were incubated at 25°C., 240rpm (50mm throw).

After 48 hours the contents were pooled and used to inoculate a further(tertiary) seed stage which comprised 100L RS1 medium+0.5 g/l NOPCOfoamaster antifoam sterilised in situ in a 150 liter fermenter for 30minutes at 121° C. The fermenter was run at 25° C., 180 rpm with anairflow of 50 l/min at 0.5 bar overpressure. Foaming was controlled bythe addition of Pluriol PE8100 antifoam obtained from BASF, Cheshire;(20% solution in soya bean oil).

1.2 Final Stage Fermentation

3000 liters of RP2 medium [Arkasoy 50, 20 g/l obtained from Arkady SoyMills, Manchester; glucose 20 g/l; L-lysine monohydrochloride, 6 g/l;bakers yeast, 6 g/l; NaCl, 5 g/l; K₂ HPO₄, 2.5 g/l; KH₂ PO₄, 2.5 g/l;MgSO₄.7H₂ O, 0.125 g/l; ZnSO₄.7H₂ O, 0.05 g/l; MnSO₄. 4H₂ O, 0.01 g/l;FeSO₄.7H₂ O, 0.02 g/l; glycerol, 30 g/l; soya bean oil, 20 g/l] togetherwith 0.5 g/l NOPCO Foamaster antifoam were sterilised in situ in a 4500liter fermenter at 121° C. for 45 minutes. After sterilisation the pHwas adjusted to pH 6.4 with 9M NH₄ OH and then inoculated with 100liters of 56 hr tertiary seed culture.

The fermenter was run at 25° C., 67 rpm (0-11 h); 75rpm (11-84 h) and 90rpm (84-202 h) with an airflow of 1500 l/min at 0.5 bar overpressure.Foaming was controlled by the addition of Pluriol PE8100 antifoam (20%solution in soya bean oil). The pH was allowed to fall naturally and nopH control was used. The fermenter was run for 158 h when 1000 liters ofbroth was removed from the vessel and the airflow was reduced to 1000l/min to maintain a constant 0.5 vvm. The fermenter was run on for afurther 44 h when the remaining 2100 liters of broth was harvested.

2.0 Isolation Procedure

2.1 Solvent Extraction

After 158 hr fermentation 1000 liters of whole broth were removed fromthe fermenter and adjusted to pH 4 with sulphuric acid. 500 liters ofMIBK was added and the mixture stirred for 2 hours.

The solvent phase was recovered using a Westfalia SA7-03-076liquid/solid centrifugal separator (Westfalia Separator Ltd; OeldeGermany). After concentration in vacuo to 20 L, the concentrate wasstored at 5° C.

After 202 hrs fermentation the remaining 2100 liters of whole broth wasremoved from the fermenter and adjusted to pH 4 with sulphuric add. 1050l of MIBK were added and the mixture stirred for 1 hour.

The solvent, phase was recovered as above and combined with the storedconcentrate from above. The combined, rich MIBK extracts wereconcentrated to give a final volume of 68 L.

2.2 Solvent Partition

125 L of methanol and 125 L of hexane were added to the concentrate andthe mixture stirred for 1/2 hour. Solvent phases were allowed toseparate by gravity overnight. The upper phase was then recovered (190L) and concentrated vacuo to give 7 kg of oil.

2.3 Initial Chromatographic Purification

The oil was loaded on a Diaion (Mitsubishi Chemical Industries Ltd TokyoJapan) HP20 column (58×30 cm) packed in 10:90 acetone-H₂ O.

After loading the column was eluted with 60:40 acetone-H₂ O. Fractionswere then taken and those containing rapamycin and 14-methylenerapamycin combined and then concentrated in vacuo to give an oil (500g). The oil was dissolved in 2 liters of acetone and stored beforesilica chromatography.

2.4 Silica chromatography

2.5 L hexane was added to the stored acetone solution and the resultloaded on a silica (Sorbsil C60 silica 40-60 μm, Rhone-Poulenc) column33×30 cm packed in 85:15, hexane-acetone. Elution was with a stepgradient of acetone in hexane. Fractions eluted with up to 70:30hexane-acetone contained 14-methylene rapamycin, these were combined andconcentrated in vacuo. 34 g of oil was obtained.

2.5 Amberchrom CG71 chromatography

2 L of Amberchrom CG71 50 μm-100 μm (Tosohaas, Stuttgart, Germany) werepacked in a medium pressure liquid chromatograph (Jobin Yvon, France, 8cm diameter) and equilibrated in hexane. After dissolving 34 g oil intoluene, elution continued at up to 8 bar pressure with 4 L of 80:20hexane-ethyl acetate followed by 7.2 L 75:25 hexane-ethyl acetate.Fractions containing 14-methylene rapamycin were combined and evaporatedin vacuo to give 1.0 g of solid.

2.6 Preparative hplc

800 mg of the above solid was dissolved in 76:24 methanol-H₂ O at up to75 mg/ml and 2 ml portions injected separately onto a reverse phase 5 μmC₁₈ column and pre column (21.4 mm×25 cm and 21.4 mm×5 cm) (RaininInstruments USA). After injection elution continued with 76:24methanol-H₂ O, at 15 ml/minute and was monitored for UV absorbance at275 nm. Fractions containing the object compound from a total of sixinjections were pooled and concentrated in vacuo to yield 83 mg of whitesolid.

Fractions containing the object compound were analysed by reverse phasehplc using a Microsorb 5 μm C₁₈ column 4.6×250 mm (Rainin InstrumentsUSA) and an Upchurch pre column (2.0×20 mm). This column was operated at30° C. and monitored by ultraviolet absorbance at 275 nm. The column waseluted with methanol-water 74:26 at 1 ml/minute. Under these conditionsthe object compound designated 14-methylene rapamycin had a retentiontime of 31.4 minutes differing from that of rapamycin.

14-methylene rapamycin was characterised by ultra violet absorbance UVmax 268,277,289 nm by mass spectroscopy FAB (M+Na)⁺ =922 and by protonand ¹³ C nuclear magnetic resonance spectroscopy.

3.0 Structural Analysis

Introduction

All NMR experiments were carried out on 18.5 mg/0.5 ml solution in CDCL₃/TMS. All measurements were carried out using a Bruker AM400spectrometer at 300K. FAB MS data was recorded on a VG ZAB massspectrometer using a matrix of 3-nitrobenzylalcohol and sodium acetate(NOBA/Na). Accurate mass measurements were carried out in duplicateusing MNa⁺ ions from rapamycin and demethoxyrapamycin as reference ions.

Results

Molecular weight determinations after accurate mass measurements were899.5739 and 899.5746. This is 14 mass units less than rapamycin(molecular formula C₅₁ H₇₉ NO₁₃) and is consistent with a formula of C₅₁H₈₁ NO₁₂ and with the presence of a methylene in place of a carbonylmoiety in the parent rapamycin.

Evidence for the structure of 14-methylene rapamycin can be found in theresults for ¹ H and ¹³ C NMR spectra given in Table 3. In particularthere is a loss of a carbonyl resonance in the ¹³ C NMR spectra and theaddition of an isolated methylene in the ¹ H and the ¹³ C NMR spectra. ACOLOC experiment positively identified the C₁₄ /H₁₄ isolated methylenesystem.

                  TABLE 3                                                         ______________________________________                                        A Table of .sup.1 H and .sup.13 C NMR Shifts (δ in ppm)                 for 14-Methylene (9-methylene)rapamycin                                       14-methylene                                                                             9-methylene                                                        rapamycin  rapamycin                                                          Atom No    Atom No      δC                                                                              δH                                      ______________________________________                                        21         1            170.64  --                                            20         2            51.56   5.51                                          19         3            27.15   2.29, 1.77                                    18         4            20.66   1.78, 1.40                                    17         5            25.33   1.79, 1.55                                    16         6            43.52   3.90, 3.38                                               7            --      --                                            15         8            173.0   --                                            14         9            38.46   2.74, 2.43                                    13         10           97.98   --                                            12         11           39.11   1.45                                          11         12           27.51   ˜1.60                                   10         13           32.28   ˜1.65                                   9          14           65.81   3.58                                          8          15           38.53   1.70, 1.55                                    7          16           84.72   3.73                                          6          17           134.35  --                                            5          18           130.55  5.96                                          4          19           126.62  6.38                                          3          20           133.50  6.37                                          2          21           130.21  6.16                                          1          22           139.83  5.59                                          33         23           34.89   2.31                                          32         24           40.36   1.51, 1.18                                    31         25           41.24   2.86                                          30         26           217.20  --                                            29         27           85.41   3.60                                          28         28           77.76   4.14                                          27         29           136.69  --                                            26         30           127.18  5.40                                          25         31           46.80   3.40                                          24         32           208.78  --                                            23         33           41.08   2.72                                          22         34           75.96   5.13                                          38         35           33.12   1.98                                          39         36           38.72   1.16, 1.05                                    40         37           33.22   1.41                                          41         38           34.09   2.13, 0.68                                    42         39           84.42   2.95                                          43         40           73.90   3.38                                          44         41           31.24   1.99, 1.34                                    45         42           31.76   1.70, 1.02                                    37         43           16.94   0.89                                          35         44           9.81    1.61                                          34         45           21.48   1.03                                          51         46           13.76   1.01                                          49         47           12.43   1.75                                          48         48           16.07   1.13                                          47         49           16.21   0.94                                          36         50           55.64   3.13                                          50         51           59.94   3.35                                          46         52           56.52   3.41                                          ______________________________________                                    

EXAMPLE 3 COMPOSITION EXAMPLES A-H A--CAPSULE COMPOSITION

A pharmaceutical composition of this invention in the form of a capsuleis prepared by filling a standard two-piece hard gelatin capsule with 50mg of a compound of the invention, in powdered form, 100 mg of lactose,32 mg of talc and 8 mg of magnesium stearate.

B--INJECTABLE PARENTERAL COMPOSITION

A pharmaceutical composition of this invention in a form suitable foradministration by injection is prepared by stirring 1.5% by weight of acompound of the invention in 10% by volume propylene glycol and water.The solution is sterilized by filtration.

C--OINTMENT COMPOSITION

Compound of the invention 1.0 g

White soft paraffin to 100.0 g

The compound of the invention is dispersed in a small volume of thevehicle and granually incorporated into the bulk of the vehicle ofproduce a smooth, homogeneous product. Collapsible metal tubes are thenfilled with the dispersion.

D--TOPICAL CREAM COMPOSITION

Compound of the invention 1.0 g

Polawax GP 200 20.0 g

Lanolin Anhydrous 2.0 g

White Beeswax 2.5 g

Methyl hydroxybenzoate 0.1 g

Distilled Water to 100.0 g

The polawax, beeswax and lanolin are heated together at 60° C. Thecompound of the invention is then added and dispersed throughout, andthe composition is allowed to cool with slow speed stirring.

E--TOPICAL LOTION COMPOSITION

Compound of the invention 1.0 g

Sorbital Monolaurate 0.6 g

Polysorbate 20 0.6 g

Cetostearyl Alcohol 1.2 g

Glycerin 6.0 g

Methyl Hydroxybenzoate 0.2 g

Purified Water B.P. to 100.00 ml

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml of thewater at 75. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75 C. and added to the aqueous solution.The resulting emultion is homogenized, allowed to cool with continuousstirring and the compound of the invention is added as a suspension inthe remaining water. The whole suspension is stirred until homogenized.

F--EYE DROP COMPOSITION

Compound of the invention 0.5 g

Methyl Hydroxybenzoate 0.0 1 g

Propyl Hydrobenzoate 0.04 g

Purified water B.P. to 100.00 ml (B.P.=British Pharmacopia)

The methyl and propyl hydroxybenzoates are dissolved in 70 ml purifiedwater at 75° C. and the resulting solution is allowed to cool. Thecompound of the invention is then added, and the solution is sterilizedby filtration through a membrane filter (0.22 mum pore size) and packedaseptically into suitable sterile containers.

G--COMPOSITION FOR ADMINISTRATION BY INHALATION

For an aerosol container by a capacity of 15-20 ml: Mix 10 mg of acompound of the invention with 0.2-0.2% of a lubricating agent, such aspolysorbate 85 or oleic acid, and disperse such mixture in a propellant,such as freon, preferably in a comination of (1,2dichlorotetrafluoroethane) and difluorochloromethane and put into anappropriate aerosol container adapter for either intranasal or oralinhalation administration.

H--COMPOSITION FOR ADMINISTRATION BY INHALATION

For an aerosol container with a capacity of 15-20 ml: Dissolve 10 mg ofa compound of the invention in ethanol (6-8 ml), add 0.1-0.2% of alubricating agent, such as polysorbate 85 or oleic acid; and dispersesuch in a propellant, such as freon, preferably a combination of (1.2dichlorotetrafluoroethane) and difluorochloromethane, and put into anappropriate aerosol container adapter for either intranasal or oralinhalation administration.

EXAMPLE 4 BIOLOGICAL EXAMPLES

The following assays were used.

Assay for Antifungal Activity

Yeast organisms (Saccharomyces cerevisiae) in logarithmic growth wereplated on complete agar medium (YPD). The compound was dissolved in anappropriate aqueous or organic solvent and placed in wells punched inthe agar. Plates were incubated for 48 hours and zones of inhibitionwere measured. The compound tested in this assay exhibited antifungalactivity.

Mitogenesis Assay for Immunosuppressive Activity

Spleen cells from BDF1 female mice were established in RPMI with 10%fetal calf serum at 5×10⁶ /ml. One hundred ml aliquots of thissuspension (5×10⁵ cells) were dispensed into 96-well round-bottomedmicrotiter plates (Linbro, Flow Laboratories). Concanavalin A (5 μg/ml)was added as the mitogenic stimulus, and the final volume in themicrotiter wells was adjusted to 200 μL with RPMI. Cell cultures wereincubated for 72 hours at 37 degrees C. in a 5% CO₂ atmosphere andpulsed with 0.5μ Ci ³ H-thymidine (specific activity 2.00 Ci/mole) forthe last 18 hour culture. The cells were harvested on an automatedmultiple sample harvester and cell-associated radioactivity counted in aBeckman liquid scintillation counter. The results are expressed as themean values derived from quadruplicate measurements. Cell viability wasdetermined by trypan blue exclusion after 72 hours of incubation.Compounds to be tested were added to the microtiter plates at theappropriate dilutions prior to the addition of cells. All of thecompounds of the invention tested in this assay exhibitedimmunosuppressive activity.

Results of these two assays, i.e., antifungal activity assay and themitogenesis assay for immunosuppressive activity, for compounds of thisinvention are provided in Table 3.

    ______________________________________                                        Yeast         Mitogenesis                                                     IC.sub.12 (nM)                                                                              IC.sub.50 (nM)                                                  ______________________________________                                        13            15                                                              ______________________________________                                    

IC₁₂.sup.(nM) refers to the concentration of drug in the antifungalassay which produces a 12 mm zone of inhibition. The aforementionedresults in the antifungal and immunosuppression assay indicate that14-methylene rapamycin has both antifungal and immunomodulatoryactivity.

While the above descriptions and Examples fully describe the inventionand the preferred embodiments thereof, it is understood that theinvention is not limited to the particular disclosed embodiments comingwithin the scope of the following claims.

I claim:
 1. A compound of formula (I): ##STR4##
 2. A compound characterised as follows:i) said compound has an apparent molecular weight of 899 by Fast Atom Bombardment (FAB) Mass spectroscopy; ii) said compound is obtainable by the cultivation of a microorganism from the genus Streptomyces; iii) said compound has 51 carbons in the molecule when subjected to ¹³ C NMR spectroscopy; iv) said compound has a characteristic UV spectrum with peaks at 268, 277 and 289 nm.
 3. A pharmaceutical composition comprising a pharmaceutically effective amount of the compound of claim 1 together with a pharmaceutically acceptable diluent or carrier. 